Storage stability is the ultimate goal for dried formulations of biological substances. Dried formulations are preferred to aqueous formulations as the water, a nucleophile in hydrolysis reactions and a plasticizer, increases the molecular mobility of reactive chemical species, making aqueous formulations of biological substances inherently less stable than their dry counterparts. This increased stability of dry formulations has focused attention on techniques of drying and led to the development of freeze-drying as a popular method of water removal. Pikal (1990a) Biopharm. 3:18-27; Pikal (1990b) Biopharm. 3:26-30; and Franks (1990) Cryoletters 11:93-100. However, despite its widespread use, many freeze-dried products are still unstable at ambient temperatures. Carpenter and Crowe (1988) Cryobiol. 25:244-255; and Crowe et al. (1990) Cryobiol. 27:219-231. Detailed theoretical analyses of the physicochemical events during freeze-drying have led to a substantial literature on the use of lyoprotectants as stabilizing excipients. Pikal (1990b); Carpenter and Crowe (1988); Crowe et al. (1990); and Levine and Slade (1988) Pure Appl. Chem. 60:1841-1864. Various carbohydrates have been advocated as stabilizing excipients in freeze-drying, and these are proposed to act via the generation of an amorphous, glassy, solid state in the freezing step. Pikal (1990b); Franks (1990); Levine and Slade (1988); and Franks et al. (1992) U.S. Pat. No. 5,098,893. Nevertheless, the freezing step remains a major variable, as evidenced by the equivocal values for the experimentally measured glass transition temperature of the maximally freeze-concentrated unfrozen matrix (T'g) for various carbohydrate excipients. Franks (1990); Levine and Slade (1988); Ablett et al. (1992) J. Chem. Soc. Faraday Trans. 88:789-794; and Roos (1993) Carbo. Res. 238:39-48.
Exposure to temperatures above the glass transition or collapse during freeze drying can result in significant activity losses. Furthermore, freezing is thought to be the major cause of protein damage during freeze-drying. Due to the loss of activity on freeze drying, recent attention has focused on the techniques of ambient temperature drying. These not only eliminate the freezing step but are more rapid and energy-efficient in the removal of water during drying. Crowe et al. (1990); Roser (1991) Biopharm. 4:47-53; Roser and Colaco (1993) New Scientist 138:24-28; Blakely et al. (1990) The Lancet 336:854-55; Colaco et al. (1990) Biotech. Intl. pp. 345-350, Century Press, London; Colaco et al. (1992) Biotech. 10:1007-1011; Franks (1991) Biopharm. 14:38-55; Franks and Hatley (1993) in "Stability and stabilization of enzymes", eds. van den Tweel et al., Elsevier, Amsterdam (1993) pp. 45-54; and Franks (1994) Bio/Tech. 12:253-256; Roser, U.S. Pat. No. 4,891,319; and Roser et al. (1991) WO91/18091.
All references cited herein, both supra and infra, are hereby incorporated herein by reference.
Stabilization of dried biological substances, particularly at ambient or higher temperatures, remains a challenge. One carbohydrate, trehalose (.alpha.-D-glucopyranosyl-.alpha.-D-glucopyranoside), has been found to be uniquely potent in prolonging shelf life of dried proteins and other biological materials for prolonged periods at ambient or higher temperatures. Stability has been assessed by recovery of biological activity upon rehydration. Roser (1991); Roser and Colaco (1993); Blakely et al. (1990); Colaco et al. (1990); Colaco et al. (1992); and Carpenter and Crowe (1988) Cryobiol. 25:459-470. Studies of other sugars, polyhydric alcohols and oligosaccharides under conditions identical to those in which trehalose provides protein stability, showed that this degree of stabilization is unique to trehalose. Some of these excipients are partially protective, however, in that they protect the biomolecules from damage during the drying process itself and confer more limited tolerance to high temperatures. Crowe et al. (1990); Roser (1991); Colaco et al. (1990); Crowe et al. (1987); Carpenter et al. (1987); and Carpenter and Crowe (1988).
There are two main hypotheses that have been postulated with respect to the molecular mechanism by which trehalose stabilizes biological molecules. Clegg (1985) in Membranes, metabolism and dry organisms, ed. Leopold, Cornell Univ. Press, Ithaca, N.Y., pp. 169-187; Burke (1985) in Membranes, metabolism and dry organisms ed. Leopold, Cornell Univ. Press, Ithaca, N.Y., pp. 358-363; Green and Angell (1989) J. Phys. Chem. 93:2280-2882; Levine and Slade (1992) Biopharm. 5:36-40; and Crowe et al. (1990) Biopharm. 6:28-37. The water replacement theory states that, being a polyol, trehalose can make multiple external hydrogen bonds which could replace the essential structural water molecules that are hydrogen-bonded to biomolecules and thus maintain their molecular structure. Clegg (1985); and Crowe et al. (1993). The glassy state theory postulates that, as the drying trehalose solutions undergo glass transformation, this results in an amorphous continuous phase in which molecular motion, and thus degradative molecular reactions, are kinetically insignificant. Burke (1985); Green and Angell (1989); and Levine and Slade (1992). Results previously obtained and those described herein are not consistent with either hypothesis being a sufficient sole explanation for the mechanism of action of trehalose.
The water replacement theory suggests that, as polyols, sugars other than trehalose should also be effective as stabilizing excipients, and, if the specific spatial combinations of hydroxyl groups are the crucial feature, then glucose should be as effective as trehalose. However, results obtained previously and presented herein show that glucose, a polyol, is among the least effective of a variety of sugars tested (see Table 1), and none of the other polyols tested was found to be as effective as trehalose (FIG. 1 and Table 1). Furthermore, if molecular mimicry of water was important, as might be expected for water replacement, then scylloinositol (with all its hydroxyl groups being axial) should, in theory, be the most effective carbohydrate, but it is among the least effective in practice. Thus, the water replacement theory cannot be a complete explanation for the mechanism of action of trehalose.
Similarly, the glassy state theory alone cannot explain the stability conferred by trehalose. In high temperature storage stability data reported in FIG. 1 above, the glass transition temperatures of the samples dried in trehalose to a water content of 2.6-3.6% were all below 37.degree. C. as measured by differential scanning calorimetry. Thus, their stability persists at well above their glass transition temperatures, and although the glassy state may be important in other systems, contrary to accepted belief (Franks et al. (1992); and Franks (1994)), it appears not to be a factor in the long-term high temperature stability of biomolecules dried in trehalose.
Although current belief is that protein stability depends almost solely on glass transition temperature, several possible contributing reactions that decrease protein stability in the presence of sugars have been proposed. Franks (1994). These possible reactions have not been analyzed and are thought to contribute little, if any, to protein instability. Franks et al. (1994); and Akers and Nail (1994) Pharm. Tech. 18:26-36.